Non-competitive immunoassay for small analytes

ABSTRACT

A non-competitive immunoassay for small analytes, wherein the analyte is reacted with two binding partners. The first binding partner binds to the analyte to form a complex between the first binding partner and the analyte, and the second binding partner binds to the complex formed by the first binding partner and the analyte. The resulting complex formed between the analyte and the binding partners is detected. The binding partners are proteins, such as antibodies including antibody fragments.

FIELD OF THE INVENTION

The present invention relates to immunoassays and especially tonon-competitive immunoassays for small analytes. The present inventionfurther relates to reagent pairs and test-kits useful in the assays, aswell as to the use of the reagent pairs and to a process for theirpreparation. Novel reagents and means for their preparation are alsoprovided.

TECHNICAL BACKGROUND OF THE INVENTION

In a competitive immunoassay, external reagent that competes with theanalyte has to be added, which is not the case in the non-competitiveassay format. The method of choice for the detection of analytes byimmuno-chemistry is nowadays a non-competitive immunoassay, where twoantibodies bind to two different epitopes of the analyte creating aso-called sandwich-type assay. Such an assay is well suited for highmolecular weight analytes and it provides improved speed, sensitivity,and specificity, which are needed in modern immunoassays. However, ithas been a difficult task to develop non-competitive assays for smallanalytes, because low molecular weight molecules are not large enoughfor binding simultaneously to more than one antibody independently.Therefore, despite many fundamental problems with respect to specificityand sensitivity, the competitive immunoassay format has been almostexclusively used for the detection of small analytes.

However, there are few publications where the development of anon-competitive immunoassay for a small analyte has been reported. Inthese papers, a secondary anti-immune complex (anti-IC) antibody, whichbinds primary anti-analyte antibody that is combined with the analytebut which does not bind the primary antibody or the analyte alone, hasbeen developed (Ullman et al., 1993; Self et al., 1994; Towbin et al.,1995). Ullman et al., 1993, describe an antibody that recognizes animmune complex of an antibody to tetrahydrocannabinol (THC). The anti-ICantibody was obtained by using an affinity labelled anti-THC antibody asimmunogen and selecting an anti-IC antibody the binding of which wasenhanced by the presence of Δ⁹THC. Self et al., 1994, used the sameprinciple in preparing anti-IC antibodies for determining digoxin.Towbin et al., 1995, report a sandwich immunoassay for the haptenangiotensin II, wherein the immunisation involves tolerization withuncomplexed primary antibody prior to immunisation with the anti-immunecomplex to obtain the anti-IC antibodies. The anti-IC antibodies used innon-competitive immunoassays for small analytes have so far beenconventional polyclonal or monoclonal antibodies obtained byimmunisation, and the assays described here include labelling of theprimary antibody and immobilisation of the secondary or vice versa.

So-called ‘idiometric’ non-competitive immunoassay for small analyteshas been developed by Mares et al., 1995. They used two types ofanti-idiotypic antibodies, which recognize different epitopes within thehypervariable region of the oeastradiol specific primary antibody. Thefirst anti-idiotypic antibody (betatype) possesses the capacity ofcompeting with the analyte for an epitope at the binding site of theprimary antibody. The second anti-idiotype (alphatype) recognizes anepitope within the variable region of the primary antibody and thebinding is not sensitive to the presence of the analyte. The alphatypeis, however, sterically hindered from binding to the primary antibody inthe presence of the betatype. These three types of antibodies permit thedevelopment of a non-competitive assay for small analytes.

So-called open sandwich immunoassays have been developed for thedetection of haptens (Suzuki et al., 2000; Suzuki et al., 1999; Yokozekiet al., 2002). They are non-competitive assays based on a phenomenonaccording to which the association of separated V_(H) and V_(L) chainsin some antibodies is strongly favoured in the presence of antigen (Uedaet al., 1996).

Despite the significant benefits of the non-competitive immunoassayformat, only few examples of that kind of assays for small analytes havebeen reported. The reason for that is most probably the difficulty ofproducing secondary (anti-immuno complex, or anti-idiotypic) antibodiesby immunising animals. For example anti-hapten monoclonal antibodies,which have been developed by hybridoma technology (Kohler and Milstein,1975), are self-antigens for mice and raising immunoresponse againstthem is difficult (Maruyama et al., 2002; Ullman et al., 1993; Kobayashiet al., 2000). A further problem is that the immune complex used forimmunisation tends to break down before the response to the immunocomplex is obtained (Ullman et al., 1993; Kobayashi et al., 2000).

The present invention now provides a non-competitive immunoassayprotocol for small analytes, which circumvents the immunisation ofanimals with the immune complex, which has been so far the mostchallenging task when anti-IC antibodies have been developed. Theinvention also facilitates a homogenous immunoassay, which furtherimproves the speed, sensitivity and simplicity of the assay.

SUMMARY OF THE INVENTION

The difficulties associated with raising anti-IC antibodies for use inimmunoassays for small analytes can now be avoided by providing thenecessary anti-IC antibodies from a display recombinant binding partnerlibrary instead of from immunised animals. A phage display antibodylibrary may be constructed, which contains a vast number of clones, fromwhich those coding the desired binding partners, such as antibodyfragments, can be enriched and selected through sequential panning. Thisprotocol opens new possibilities for developing rapid, reliable andsimple immunoassays for small analytes in a cost-effective and feasibleway.

Consequently, one object of the present invention is a non-competitiveimmunoassay for a small analyte comprising reacting a sample containingsaid analyte with a reagent pair comprising a first binding partner thatbinds to said analyte, and a second binding partner that binds to thecomplex of said analyte and said first binding partner. The immunoassayis characterized in that the second binding partner is obtained from adisplay recombinant binding partner library by selecting a bindingpartner that binds to said complex of the analyte and first bindingpartner, and determining the binding of the second binding partner thusindicating the presence of the analyte in the sample.

Another object of the invention is a reagent pair for a non-competitiveimmunoassay for a small analyte, comprising a first binding partner thatbinds to said analyte, and a second binding partner that binds to thecomplex of said analyte and said first binding partner, characterized inthat the second binding partner is obtained from a display recombinantbinding partner library by selecting a binding partner that binds tosaid complex of the analyte and first binding partner.

Still another object of the present invention is a test kit for anon-competitive immunoassay for a small analyte, said kit comprising areagent pair comprising a first binding partner that binds to saidanalyte, and a second binding partner that binds to the complex of saidanalyte and said first binding partner, characterized in that the secondbinding partner is obtained from a display recombinant binding partnerlibrary by selecting a binding partner that binds to said complex of theanalyte and first binding partner.

The invention is also directed to the use of a reagent pair comprising afirst binding partner that binds to an analyte, and a second bindingpartner that binds to the complex of said analyte and said first bindingpartner, in a non-competitive immunoassay for a small analyte, wherebythe second binding partner is obtained from a display recombinantbinding partner library by selecting a binding partner that binds tosaid complex of the analyte and first binding partner.

A still further object of the invention is a process for preparing areagent pair for a non-competitive immunoassay for a small analytecomprising providing a first binding partner that binds to said analyte,and a second binding partner that binds to the complex of said analyteand said first binding partner, characterized in that the second bindingpartner is obtained from a display recombinant binding partner libraryby selecting a binding partner that binds to said complex of the analyteand first binding partner.

The invention also provides novel recombinant binding proteins,characterized in that they comprise the ligand-binding portion of M1 Fabcomprising SEQ ID NO 1 and SEQ ID NO 2; M2 Fab comprising SEQ ID NO 3and SEQ ID NO 4; or K11 scFv comprising SEQ ID NO 5.

DNA encoding the novel binding proteins as well as host cells expressingthem are also included in the invention.

Advantagous embodiments of the invention are set forth in the dependentclaims.

Other objects, details and advantages of the present invention willbecome apparent from the following drawings, detailed description andexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A competitive ELISA using M1 Fab. Urine+ is urine spiked with 1μg/ml of morphine.

FIG. 2. A non-competitive time resolved fluorescent immunoassay (TR-FIA)with the Eu labelled anti-M1+ morphine immune complex scFv fragment K11.a) Cross-reactivity and sensitivity of the assay. b) Sensitivity of theassay with S1 urine control dilutions as samples.

FIG. 3. A homogenous TR-FRET immunoassay of morphine.

FIG. 4. Analysis of cross-reactivity of M1 anti-morphine Fab-fragmentwith codeine, heroin, noscapine and papaverine by a competitive ELISA.

FIG. 5. A comparative TR-FRET based homogeneous immunoassay formorphine.

DETAILED DESCRIPTION OF THE INVENTION

The reagent pair for the non-competitive immunoassay of the inventioncomprises a first binding partner and a second binding partner. Thefirst binding partner binds to the analyte to form a complex between thefirst binding partner and the analyte. The second binding partner bindsto the complex formed by the first binding partner and the analyte. Thebinding partners are usually proteins such as antibodies includingantibody fragments that have the desired binding properties. An antibodyis an immunoglobulin molecule and it can belong to any of classes IgG,IgM, IgE, IgA or IgD; IgG and IgM being the most frequently used.Preferably the binding partners are antibody fragments comprising theligand-binding site, such as Fab, or scFv fragments. The fragment knownas the Fab fragment (fragment antigen binding) consists of the variableand constant domain of an immunoglobulin light chain covalently attachedby a disulfide bridge to the variable and first constant domain of animmunoglobulin heavy chain. Fv (variable domain) means the variableregions of the immunoglobulin molecule that are responsible for theligand binding. ScFv (single chain Fv) means a molecule wherein thevariable domains of the heavy and light chain of an antibody are linkedby a peptide to form a single polypeptide chain synthesized from asingle mRNA molecule. The variable regions of an immunoglobulin heavychain and light chain are together responsible for the ligand binding.Ligand is the substance to which the binding partner binds, inconnection with antibodies it is an antigen or a hapten.

The first binding partner may be a conventional polyclonal or monoclonalantibody or fragment thereof, but preferably it is a recombinant one, asis the second binding partner. When the first binding partner has beenselected for, it is complexed with its ligand and this complex is usedto select for the second binding partner from a recombinant library. Thefirst binding partner without the ligand is used as contraselection. Thesecond binding partner should only recognise complexes, not free firstbinding partner nor free antigen to any significant extent.

The recombinant binding partner library is conveniently an expressionlibrary, which is typically a display library. The general principle ofthe display recombinant binding partner libraries is that they presentthe binding partner as a fusion protein on the surface, which may be thesurface of a microbial cell such as a yeast or bacterial cell, or aphage. The display recombinant binding partner library can also be adisplay library, where stable complexes of nascent protein and mRNA areproduced in an in vitro expression system. Phage display libraries arethe most frequently used. Antibody phage display technology and itsapplications are described e.g. in Hoogenboom et al., 1998.

A phage display antibody library may be constructed by cloningimmunoglobulin domains coding cDNAs into an appropriate phage displayvector. DNA encoding for millions of variants of antibody fragments isbatch-cloned into the vector as part of the phage coat protein. Largelibraries containing millions of antibody fragments with differentspecificities can be obtained by transforming the vectors in bacteria.Cultivation of the bacteria leads to the expression of phages displayingantibody fragments on their surface. The gene for the displayed antibodyis carried in the phage genome, thus linking genotype with phenotype.The physical linkage between the displayed protein and its DNA allowsscreening of vast numbers of variants of the protein, each linked to itscorresponding DNA, by a simple in vitro selection procedure calledpanning. In its simplest form, panning is carried out by incubating thepool of phage-displayed variants with the ligand of interest that hasbeen immobilized on a carrier, washing away unbound phage, and elutingspecifically bound phage by disrupting the binding to the ligand. Theeluted phage is then amplified in vivo. The process is repeated severaltimes, resulting in stepwise enrichment of the phage pool in favour ofthe tightest binding sequences. After about 3 to 6 rounds of selectionand amplification, the best clones are sequenced and transformed into ahost cell for further expression. The host cell may be a eucaryotic orprocaryotic cell e.g. a yeast, animal, plant or insect cell or bacterialcell. It may even be a hybridoma cell, which after transformationproduces a recombinant monoclonal antibody. The recombinant bindingpartner or at least part of it may also be produced synthetically.

The concept to use recombinant antibody libraries makes thenon-competitive sandwich assay for small analytes feasible. The sandwichcan be detected by all the standard immunoassays. Usually one partner isimmobilized on a carrier, such as a microtiter well or a bead. Asandwich is formed in the presence of analyte and the other bindingpartner. The sandwich may be detected e.g. by using secondary antibodiesor by labelling at least one of the binding partners. The label can beany conventional label, such as a radioactive label, an enzyme, or afluorescent compound. The assay can be e.g. ELISA or FIA.

A great advantage of the reagent pair of the present invention is thatit enables a homogenous non-competitive immunoassay, i.e. an immunoassaythat is carried out in solution. The avoidance of immobilising andwashing steps makes the assay extremely simple. Such a test is alsosuitable for testing on-site i.e. in places elsewhere than thelaboratory.

A preferred homogenous immunoassay is one based on fluorescenceresonance energy transfer (FRET), for review see Szollosi et al., 1998.In FRET, energy from a molecular fluorophore (donor) is excited to ahigh-energy state and transferred to another fluorophore (acceptor) viaintermolecular dipole-dipole coupling. This is possible only if thedistance between the donor and the acceptor is short (10-100 Å) and thefluorescence spectrum of the donor and the absorption spectrum of theacceptor partially overlap. The energy transfer is then detected as achange in fluorescence. Often time-resolved fluorescence is utilized(Hemmila et al., 1988).

FRET is applied to the present invention by labelling the two bindingpartners, which preferably are antibody fragments, with fluorophoresthat form a FRET donor-acceptor pair. When the binding partners and theanalyte are small the fluorophores come into very close proximity, and ameasurable FRET signal is obtained.

The invention provides a convenient and rapid analytical tool for lowmolecular weight analytes, such as therapeutic and abused drugs,steroids, hormones, metabolites, and environmental pollutants andtoxins. A common feature of these small analytes is that they are toosmall for conventional sandwich assays where two antibodies recognizingdifferent epitopes of the antigen are used. The molecular weight ofthese small analytes are normally less than 5000, but the limits are notabsolute.

The immunoassay may be employed in all kinds of investigations, such asin detecting environmental hazards, toxic compounds in food and feed,chemicals indicative of ongoing processes e.g. of microbial processes inbuildings, metabolic processes of living organisms, and in clinicaltests, drug monitoring and pharmacological research. The assays areextremely suitable for detecting drugs of abuse, such as opiates (e.g.morphine), amphetamines, cannabinoids (e.g. tetrahydrocannabinol (THC)),barbiturates, benzodiazepines, cocaine, LSD, methadone, methaqualone,phencyclidine, propoxyphene, tricyclic antidepressants. The homogenousassay provides an excellent and convenient tool for on-site tests e.g.to be used by the police in raiding drivers etc. The sample to beanalysed for e.g. drugs and abused drugs may be any body fluid sample,such as blood, serum, urine or saliva.

The reagent pair of the invention may be included in a test-kit. Thistest-kit may further comprise any other reagents needed for the assay,such as reaction solutions, buffers, washing solutions and detectingmeans, such as labels and optionally a fluorometer. Preferably thetest-kit comprises multiple reagent pairs physically separated from eachother, e.g. many in the form of a microarray, whereby e.g. manydifferent drugs of abuse may be tested simultaneously from a singlesaliva sample.

In a special embodiment of the invention, a reagent pair for detectingmorphine is produced and employed in a homogenous immunoassay for saidsubstance. The first binding partner is a Fab fragment obtained from aphage display antibody library produced from cDNA from a mouse immunizedwith morphine conjugated to an immunocarrier BSA. Morphine specificantibody phages were enriched by selecting those binding to morphineconjugated BSA and sorting out those binding to BSA alone. After severalpanning rounds two high binding clones are sequenced and expressed. Theexpressed Fab fragments were named M1 Fab and M2 Fab.

The second binding partner is obtained from a naive scFv antibodyfragment phage display library by selecting antibodies that bind to acomplex of morphine and M1 Fab. First the phages are preincubated tobound M1 Fab to sort out those binding to M1 Fab as such. The unboundphages are separated and incubated with a mixture of morphine andimmobilised M1 Fab to select the phages that bind to the immunocomplexformed between the immobilized M1 Fab and morphine. Unbound phages arewashed away, and then those bound to the complex are eluted. Thebackground is monitored by checking the binding to M1 Fab in the absenceof morphine. After several panning rounds a number of clones are pickedup, sequenced and expressed resulting in scFv fragment K11.

A fluorescence-based immunoassay is performed using M1 Fab labelled witheuropium as a first binding partner, and scFv fragment K11 labelled withCy5 as a second binding partner. The binding partners are incubated withsaliva or urine samples containing morphine and then fluorescence ismeasured after a predetermined time. The assay is completely homogeneousand the signal is readable in about 5 min. The sensitivity for bothurine and saliva is clearly higher than that demanded by the authoritiesin the case of morphine, our model analyte. The performance of the testis such that the reagents are in the well of a microtiter plate anddilution series of either saliva or urine is added. In a preferred modefor e.g. police field use, the reagents are in dry form in a vessel.Saliva is added, which dissolves the reagents and the result can be readwithout further processes.

The novel recombinant binding proteins provided by the inventioncomprise any ligand-binding portion of M1 Fab, M2 Fab or K11 scFv. By“ligand-binding portion” is meant that part of the molecule that isresponsible for the binding. Minor variations or modifications of thesequences set forth in the description and claims are still within thescope of the invention provided that they do not affect the bindingactivity of the proteins.

The invention is illustrated by the following non-limiting example. Itshould be understood, however, that the embodiments given in thedescription above and in the examples are for illustrative purposesonly, and that various changes and modifications are possible within thescope of the invention.

EXAMPLE 1

Development of an Anti-Morphine Antibody

Immunisation of Mice

Four six-week-old female Balb/c mice were immunised in three-weekintervals with morphine conjugated BSA (Fitzgerald) in Freund'sadjuvant. Serum samples were tested after second booster and the mouseshowing the best response against the antigen in direct ELISA wasselected to be the source of an antibody phage display library.

Construction of the Antibody Phage Display Library

All basic recombinant DNA methods were performed essentially asdescribed (Sambrook et al. 1990). The mouse with the highest antibodyresponse to morphine-BSA conjugate was sacrificed and the total RNA wasisolated from the spleen cells using the RNagentse Total RNA IsolationSystem (Promega Co., WI, USA). The mRNA pool of the total RNA wasisolated with the Oligotex mRNA Kit (QIAGEN Inc., Germany). The cDNA wassynthesised from the mRNA with oligo-dT priming. Genes encoding antibodyFab fragments were amplified with PCR using antibody kappa light chainand heavy chain variable region and constant region specific primers.Antibody light chain PCR products were pooled and digested with Nhel andAscI restriction enzymes, purified by preparative agarose gel incombination with the QIAquick Gel Extraction Kit (QIAGEN Inc., Germany).The agarose gel purified antibody light chain DNA was ligated into theFab phagemid vector phagemid9 derived from pComb3 (Barbas et al., 1991)and transformed into the E.coli XL1-Blue strain (Stratagene) byelectroporation. Plasmid DNA was isolated with the QIAGEN Plasmid MidiKit (QIAGEN Inc., Germany) from the overnight culture. PCR productsencoding the Fd region (variable and first constant region) of the heavychain were pooled and digested with Sfil and Notl restriction enzymes,purified by preparative agarose gel isolation and ligated to thephagemid vector containing the light chain DNA. The phagemid vectorencoding both the heavy and light chain of the Fab fragment wastransformed into the E.coli TOP10F′ bacteria (Invitrogen Inc., CA, USA)by electroporation. Transformed bacteria were incubated over night at+37° C. on a shaker and plasmid DNA was isolated with the QIAGEN PlasmidMidi Kit. The diversity of the antibody library was ensured bysequencing partial V_(L)-or V_(H)-gene regions of individual clones.

A phage display antibody library was made as follows: 4 μg of antibodylibrary plasmid DNA was transformed into Ecoli TOP10F′ byelectroporation in two parallel transformations. After transformations,cells were suspended into 2.8 ml of SOC medium and incubated for 1 h at+35° C. on a shaker. 7 ml of prewarmed (+37° C.) SB medium, 20 μg/ml ofcarbenicillin, and 10 μg/ml of tetracycline was added. After 1-hincubation at +35° C. on a shaker, 30 μg/ml of carbenicillin was addedand the incubation was continued for 1 h after which 1 ml (˜10¹¹ pfu) ofhelper phage VCS-M13 (Stratagene) was added and the phages were let toinfect the bacteria for 20 min at +35° C. with a slow shaking. Theparallel transformations were joined together and 80 ml of pre-warmed(+37° C.) SB medium with 50 μg/ml carbenicillin and 10 μg/ml oftetra-cycline was added. After 2-h incubation at +35° C. on a shaker, 70μg/ml of kanamycin was added and the incubation was continued overnight.

Cells were centrifuged for 15 min at 4000 g at +4° C. 20 ml of 20% PEG,2.5 M NaCl (PEG/NaCl) was added to the supernatant and it was incubatedfor 30 min on ice. PEG precipitated phages were centrifuged for 20 minat 13000 g at +4° C. The pellet was suspended in 2 ml of PBS and 1 mlwas transferred into two eppendorf tubes. After centrifugation for 5 minat +4° C., phages were precipitated by adding 200 μl of PEG/NaCl to thesupernatant. The solution was mixed and centrifuged for 5 min at +4° C.The pellet was suspended in 1 ml of PBS or PBS+1% BSA. 1 μl of 20%Na-azide was added as a preservative. The phage display antibody librarywas stored at +4° C.

Selection of the Anti-Morphine Library

Morphine specific antibodies were selected from the phage displayantibody library by the following selection procedure: A microtiter wellwas coated over night at +4° C. with 1 μg of morphine conjugated BSA(morphine-BSA; Fitzgerald Industries International, Inc., MA, USA) in100 μl of 100 mM Na-bicarbonate buffer, pH 9.8. The well was washed twotimes with PBS and blocked with 1% BSA in PBS for 1 h at +37° C. 100 μlof the phage library was incubated in the well for 1 h at RT withshaking, after which the unbound phages were removed and the well waswashed 22 times with PBS. Bound phages were eluted with 100 μl of 100 mMHCl (pH 2.2) for 15 min. Eluted phages were removed from the well andneutralised with 1 M Tris. 3 ml of fresh E.coli XL1-Blue cells (OD₆₀₀≈1)grown in SB supplemented with 10 μg/ml of tetracycline was infected witheluted phages at +35° C. for 15 min. 7 ml of prewarmed SB (+37° C.) with20 μg/ml of carbenicillin and 10 μg/ml of tetracycline was added and theculture was incubated for 1 h at +35° C. on a shaker. 30 μg/ml ofcarbenicillin was added and incubation was continued for 1 h. 1 ml ofhelper phage VCS-M13 was added to the culture and incubated at +35° C.for 15 min with a slow shaking. The culture was diluted with 90 ml ofprewarmed (+37° C.) SB with 50 μg/ml carbenicillin and 10 μg/mltetracycline. After 2 h incubation at +35° C. on a shaker, 70 μg/ml ofkanamycin was added and the incubation was continued over night. Thepurification of the amplified phages was performed by PEG precipitationas described above.

The purified phages were used in further enrichment rounds. After fourselection rounds, morphine specific antibody phages had been enrichedover 1000 times when compared to the background. The background bindingof the phages was monitored in parallel in each selection round byincubating the phage library in a BSA coated microtiter well. The wellwas washed and the phages were eluted as in the morphine-BSA coatedwell. The enrichment of the morphine specific phages was monitored bycomparing the amount of the eluted phages from the morphine-BSA coatedwell to the amount of eluted phages from the background well.

Characterisation of Individual Clones

After the fourth panning round, the phagemid DNA was isolated with theQIAGEN Plasmid Midi Kit. The plasmid was digested with NheI and NotIrestriction enzymes to isolate the Fab gene fragment. The agarose gelisolated DNA was ligated to the expression vector pKKtac. The ligationreaction was transformed into the E.coli XL1-Blue cells.

Individual clones were picked and miniprep DNA was extracted with theQIAprep Spin Miniprep Kit (QIAGEN Inc., Germany). According tosequencing of the minipreps, two different Fab clones were found. Theseclones were named M1 and M2. The amino acid sequences of the M1 Fabfragment were SEQ ID NO 1 (light chain) and SEQ ID NO 2 (heavy chain).Amino acids no. 3 to 108 represent the variable region and no. 109 to215 the constant region of the M1 light chain (SEQ ID NO 1). Amino acidsno 4 to 123 represent the variable region and no. 124 to 226 theconstant region of the M1 heavy chain (SEQ ID NO 2). The amino acidsequences of M2 Fab fragment were SEQ ID NO 3 (light chain) and SEQ IDNO 4 (heavy chain). Amino acids no. 3 to 108 represent the variableregion and no. 109 to 215 the constant region of the M2 light chain (SEQID NO 3). Amino acids no. 4 to 123 represent the variable region and no.124 to 226 the constant region of the M2 heavy chain (SEQ ID NO 4). Therest of the amino acids derive from the cloning technique, and some ofthe C-terminal amino acids facilitate the isolation and purification ofthe protein. Small-scale (3 ml) Fab expression cultures were also made.Periplasmic fraction of the cells was isolated by freezing and thawingthe cells for three times in PBS.

The binding of individual Fab clones to morphine was tested by ELISA inmorphine-BSA coated microtiter wells: Microtiter wells were coated with200 ng of morphine-BSA in 0.1 M Na-bicarbonate buffer, pH 9.8 for overnight at +4° C. Control wells were filled only with the buffer. Aftercoating, wells were washed three times with PBS and blocked with 0.5%BSA in PBS (BSA/PBS) for 1 h at RT. Wells were washed three times withPBS and 1:10 dilution of the periplasmic fractions was added into wellsin 100 μl of BSA/PBS. After 1-h incubation at RT on a shaker, wells werewashed three times with PBS and 1:2000 diluted alkaline phosphataseconjugated anti-mouse Fab specific antibody (Sigma, A-1293) was added in100 μl of BSA/PBS. The wells were incubated for 1 h at RT on a shakerand washed three times with PBS. 100 μl of alkaline phosphatasesubstrate solution (2 mg/ml of p-nitrophenylphosphate di-Na-salt indiethanolamine-MgCl-buffer) was added to the wells and absorbance wasmeasured. According to the preliminary results, the M1 Fab showed betterperformance in the assay and was therefore chosen for the continuation.

Fermentation and Purification

The anti-morphine Fab fragment M1 in the expression vector pKKtac wastransformed in the E.coli expression strain RV308. The Fab was expressedby fed-batch fermentation in a Bio-Flow IV fermenter (New Brunswick) andpurified by the Sepharose SP ion-exchange and protein G chromatography(Pharmacia).

Performance of the Primary Antibody in a Competitive Immunoassay

The performance of the M1 anti-morphine Fab fragment was tested in acompetitive ELISA assay using the commercial urine toxicology controlsS1 and S3 (Bio-Rad). The S1 urine toxicology control contains drugs anddrug metabolites (including morphine) at concentrations 20-25% belowimmunoassay cut-off levels as recommended by the U.S. Substance Abuseand Mental Health Services Administration (SAMSHA) and other agencies.The S3 urine toxicology control contains drugs and drug metabolites atconcentrations approximately three times immunoassay cut-off levels.Microtiter wells were coated over night at +4° C. with 500 ng ofmorphine-BSA in 100 μl of Nabicarbonate buffer, pH 9.8. Wells werewashed three times with PBS and blocked with BSA/PBS for 1 h at RT.After three washes with PBS, S1, S3 (Bio-Rad), positive control, ornegative control urine dilutions were added to the wells in 100 μl ofBSA/PBS spiked with 1 ng of M1 Fab. Wells were incubated for 2 h at RTon a shaker and washed three times with PBS. 1:2000 diluted alkalinephosphatase conjugated anti-Fab antibody (Sigma, A-1293) was added tothe wells in 100 μl of BSA/PBS and incubated for 1 h at RT on a shaker.Wells were washed three times with PBS, 100 μl of alkaline phosphatasesubstrate solution was added and A₄₀₅ was measured. Results are shown inFIG. 1. A positive result could be achieved with 1:64 dilution of thesample.

Development of an Anti-Immune Complex Antibody Specific to the ImmuneComplex of M1 Fab and Morphine

Selection of the Immune Complex Specific Antibody from a Naive HumanscFv Phage Display Library

M1 Fab fragment was biotinylated with ImmunoPure Sulfo-NHS-LC-Biotin Kit(Pierce). Biotinylated antibody was purified and buffer was changed toPBS with Econo-Pac 10DG Columns (Bio-Rad, CA, USA). 200 μl of a naivehuman scFv phage display library (Kappa or Lambda light chain) inBSA/PBS was preincubated with 10 μl of streptavidin coated magneticbeads (Dynal, M-280) and 0.5 μg of biotinylated M1 Fab for over night at+4° C. The naive human scFv phage display library was constructed frompooled lymphocytes of 50 healthy individuals. The size of the librarywas estimated to be 1×10⁸ clones. The naive human scFv phage displaylibrary contains the IgM specific V_(H)-genes combined either with thekappa or lambda specific V_(L)-genes. Unbound phages were separated fromthe beads and 100 μl of them was incubated with 100 ng of morphine, 500ng of biotinylated M1 Fab, and 5 μl of streptavidin coated magneticbeads for 1 h at RT on a shaker. The background for the selectionprocedure was implemented in a similar way but omitting the morphinefrom the binding reaction. Magnetic beads were washed five times with0.5 ml of PBS and bound phages were eluted with 100 μl of HCl (pH 2.2)for 30 min. The eluted phages were neutralised with 1M Tris and E.coliXL1-Blue cells were infected. Cells were grown and phages were purifiedas described previously. After five panning rounds enrichment was seenwith the scFv library having the kappa light chains, when the amount ofeluted phages was compared to the amount of eluted phages from thebackground control well. The enrichment of specific binders to theimmune complex, formed by M1 Fab and morphine, was also clearly seen ina phage ELISA when the eluted phage pools from the selection rounds weretested.

Characterisation of Individual Clones

Individual phage clones were picked and they were sequenced. All of theclones had the same sequence (SEQ ID NO 5). The amino acid sequence ofanti-M1+morphine immune complex scFv fragment was named K11 scFv. Aminoacids no. 3 to 120 represent the heavy chain variable region, no. 140 to246 represent the light chain variable region, and no. 121 to 139represent the linker of K11 scFv (SEQ ID NO 5). The rest of the aminoacids derive from the cloning technique, and some of the C-terminalamino acids facilitate the isolation and purification of the protein.

Expression and Purification

The gene encoding the scFv fragment K 11 was inserted into the 5expression vector pKKtac and transformed into the E.coli RV308 strain.Cells were inoculated into 20 ml of LB with 100 μg/ml ampicillin andwere incubated over night at +37° C. on a shaker. From the overnightculture a 10 ml inoculate was added into two erlenmeyer bottlescontaining 500 ml of LB with 100 μg/ml of ampicillin. Cells wereincubated at +37° C. on a shaker until the OD₆₀₀ was 1 after which 0.5ml of 1M IPTG and 100 μg/ml of ampicillin were added into the cultureand the incubation was continued over night at +30° C. on a shaker. Boththe supernatant of the culture medium and the periplasmic fraction ofthe cells were used for the purification of the K11 scFv. Cells werecentrifuged at 4000 g for 15 min and the supernatant was poured into aclean flask. The cells 1.5 were resuspended in 20 ml of PBS. Theperiplasmic fraction of the cells was isolated by freezing (−70° C.) andthawing (+37° C.) the cells for three times. After centrifugation at12000 g for 30 min, the clear periplasmic supernatant was taken. Theculture medium and the periplasmic fraction were treated with 2 mg/ml ofDNasel to remove residual chromosomal DNA for two hours at +37° C. Sincethe expressed K11 scFv has a 6× His-tag in its C-terminus, it could bepurified by immobilised metal affinity chromatography (IMAC). The K11scFv was purified by expanded bed chromatography using STREAMLINEChelating (Amershampharmacia biotech) as a matrix and copper as themetal chelate. The purity of K11 scFv was checked with SDS-PAGE.

Development of a Non-Competitive Immunoassay for Morphine

Labelling of the Anti-Immune Complex Antibody K11 scFv with Europium

The purified anti-M1 and morphine immune complex scFv fragment K11 waslabelled with europium chelate by the DELFIA Eu-Labelling Kit (Wallac)as described by the manufacturer. Buffer of the labelled K11 scFv waschanged to 50 mM Tris pH 7.8, 0.9% NaCl. Labelling yield was 0.4Eu/scFv.

A Non-Competitive Immunoassay for Morphine

The sensitivity and cross-reactivity of K11 scFv was studied by anon-competitive immunoassay. Transparent Streptawell (Roche)streptavidin coated microtiter wells were washed three times with PBS.500 ng of biotinylated anti-morphine M1 Fab was added into the wells in100 μl of BSA/PBS and the wells were incubated for 30 min at RT on ashaker. After three PBS washes, sample dilutions spiked with morphine,amphetamine, tetrahydrocannabinol (THC) or S1 urine control were addedinto the wells in 50 μl of BSA/PBS. 1:100 diluted europium labelled K11scFv was added into the wells in 50 μl of BSA/PBS and the incubation wascontinued for 1 h. Wells were washed three times with PBS and 100 μl ofEnhancement solution (Wallac) was added into the wells. After 15-minshaking at RT, fluorescence was detected by VictorV fluorometer(Wallac). Results are shown in FIG. 2. There is a significant differencein affinity between the binding of K11 scFv to the primary antibodyfragment M1 and to the M1 and morphine immune complex. This allows thedetection of 1 ng/ml of morphine in a sample. No cross-reactivity withamphetamine or THC was detected.

A Homogenous Time Resolved Fluorescent Resonance Energy Transfer(TR-FRET)

The anti-morphine Fab fragment M1 was labelled with europium by theLANCE Eu-W1024 ITC chelate Kit (Wallac). The buffer of the labelled M1was changed to 50 mM Tris, pH 7.8, 0.9% NaCl. The labelling yield was1.1 Eu/Fab. The anti-M1+morphine immune complex scFv fragment K11 scFvwas labelled with Cy5 by the FluoroLink-Ab Cy5 labelling kit(Amershampharmacia biotech). The labelling yield was 2 Cy5/scFv.

Saliva was spiked with various dilutions of morphine and these sampleswere filtered through cotton wool before adding into the blackmicrotiter wells (Nunc). 1 μg of europium labelled M1 Fab and 1 μg ofCy5 labelled K11 scFv was added into the wells in 50 μl of BSA/PBS.TR-FRET was read after 5, 15, 30, or 60 min incubation at RT by VictorVfluorometer (Wallac). Results are shown in FIG. 3. After five minutesincubation, 1 ng/ml of morphine in saliva was detected.

While one strategy for providing reagents and assays of the inventionhas been described, numerous variations and modifications will becomeapparent to the person skilled in the art.

EXAMPLE 2

Cross-Reactivity of the M1 Fab-Fragment with Codeine, Heroin, Noscapineand Papaverine

Cross-reactivity of M1 anti-morphine Fab-fragment with codeine, heroin,noscapine and papaverine was tested in a competitive ELISA. Microtiterplate wells were coated o/n at +4° C. with 500 ng of morphine-BSA in 100μl of Na-bicarbonate buffer, pH 9.8. The wells were washed three timeswith PBS and blocked with 0.5% BSA/PBS for 1 h at RT and washed againthree times with PBS. Two parallel 100 μl samples containing morphine,codeine, heroin, noscapine or papaverine (39, 78, 156, 313, 625, 1250,2500 or 5000 ng/ml) in PBS with 5 ng of purified M1 Fab were added intothe wells and incubated for 30 min at RT on a shaker and washed threetimes with PBS. 1:2000 diluted alkaline phosphatase conjugated anti-Fabantibody (Sigma, A-1293) was added to the wells in 100 μl of 0.5%BSA/PBS and incubated for 30 min at RT on a shaker. Wells were washedthree times with PBS, 100 μl of alkaline phosphatase substrate solutionwas added and A₄₀₅ was measured. The results are shown in FIG. 4.According to the competitive ELISA result M1 antibody has highcross-reactivity to codeine and heroin, which are structurally verysimilar with morphine.

A Homogenous Time Resolved Fluorescent Resonance Energy Transfer(TR-FRET) Imunoassay for Morphine

The anti-morphine Fab fragment M1 was labelled with europium by LANCEEu-W1024 ITC chelate Kit (Wallac). The buffer of the labelled M1 waschanged to 50 mM Tris, pH 7.8, 0.9% NaCl. The labelling yield was 1.1Eu/Fab. The anti-M1+morphine immune complex scFv fragment K11 waslabelled with Cy5 by FluoroLink-Ab Cy5 labelling kit (Amersham PharmaciaBiotech). The labelling yield was 2 Cy5/scFv.

Saliva was spiked with a high concentration (10 μg/ml) of the followingdrugs: morphine, heroin, codeine, papaverine and noscapine. The sampleswere filtered through cotton wool before adding into black microtiterwells (Nunc). 1 μg of europium labelled M1 Fab and 1 μg of Cy5 labelledK11 scFv was added into the wells in 50 μl of BSA/PBS. As the controlthe background fluorescence from the M1 and K11 antibody pair withoutany added drug was measured. TR-FRET was read after 5 min incubation atRT by VictorV fluorometer (Wallac). The results are shown in FIG. 5.

Saliva sample spiked with morphine is giving a high fluorescent value,whereas the other drugs are giving fluorescent values that are similarto the background fluorescence detected from the control (labelled M1and K11 scFv fragments without any added drug). K11 scFv binds theimmune complex formed between M1 and mophine with extremely highspecificity. K11 is able to discriminate completely M1 and morphineimmune complex from the immune complexes between M1 and heroin or M1 andcodeine, which gave fluorescent values corresponding to the backgroundlevel.

REFERENCES

Barbas, C. F. III, Kang, A. S., Lerner, R. A., Benkovic, S. J. (1991)Assembly of combinatorial antibody libraries on phage surfaces: the geneIII site. Proc. Natl. Acad. Sci. USA 88:7978-7982.

Hemmila, I., Malminen, O., Mikola, H., Lbvgren, T. (1988) Homogeneoustime-resolved fluoroimmunoassay of thyroxin in serum. Clin. Chem.34:2320-2322.

Hoogenboom H. R., de Bruïne, A. P., Hufton, S. E., Hoet, R. M., Arends,J.-W., and Roovers, R. C. (1998) Review article: Antibody phage displaytechnology and its applications. Immunotechnology 4, 1-20.

Kobayashi, N., Oiwa, H., Kubota, K., Sakoda, S., Goto, J. (2000)Monoclonal antibodies generated against an affinity-labeled immunecomplex of an anti-bile acid metabolite antibody: an approach tononcompetitive hapten immunoassays based on anti-idiotype oranti-metatype antibodies. J Immunol Methods, 245, 95-108.

Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cellssecreting antibody of predefined specificity. Nature, 256, 495-7.

Mares, A., De Boever, J., Osher, J., Quiroga, S., Barnard, G. and Kohen,F. (1995) A direct non-competitive idiometric enzyme immunoassay forserum oestradiol. J Immunol Methods, 181, 83-90.

Maruyama, H., Sperlagh, M., Zaloudik, J., Liang, S., Mizuki, K.,Molthoff, C. and Herlyn, D. (2002) Immunization procedures foranti-idiotypic antibody induction in mice and rats. J Immunol Methods,264, 121.

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1990) Molecular Cloning:A laboratory Manual, 2nd Ed., Cold Spring Horbor Laboratory Press, ColdSpring Harbor, NY.

Self, C. H., Dessi, J. L. and Winger, L. A. (1994) High-performanceassays of small molecules: enhanced sensitivity, rapidity, andconvenience demonstrated with a noncompetitive immunometric anti-immunecomplex assay system for digoxin. Clin Chem, 40, 2035-41.

Suzuki, C., Ueda, H., Mahoney, W. and Nagamune, T. (2000) Open sandwichenzyme-linked immunosorbent assay for the quantitation of small haptens.Anal Biochem, 286, 238-46.

Suzuki, C., Ueda, H., Tsumoto, K., Mahoney, W. C., Kumagai, I. andNagamune, T. (1999) Open sandwich ELISA with V(H)-/V(L)-alkalinephosphatase fusion proteins. J Immunol Methods, 224,171-84.

Szöllösi, J., Damjanovich, S., Mátyus, L. (1998) Application ofFluorescence Resonance Energy Transfer in the Clinical Laboratory:Routine and Research. Communications in Clinical Cytometry, 34:159-179.

Towbin, H., Motz, J., Oroszlan, P. and Zingel, O. (1995) Sandwichimmunoassay for the hapten angiotensin II. A novel assay principle basedon antibodies against immune complexes. J Immunol Methods, 181, 167-76.

Ueda, H., Tsumoto, K., Kubota, K., Suzuki, E., Nagamune, T., Nishimura,H., Schueler, P. A., Winter, G., Kumagai, I. and Mohoney, W. C. (1996)Open sandwich ELISA: a novel immunoassay based on the interchaininteraction of antibody variable region. Nat Biotechnol, 14,1714-8.

Ullman, E. F., Milburn, G., Jelesko, J., Radika, K., Pirio, M., Kempe,T. and Skold, C. (1993) Anti-immune complex antibodies enhance affinityand specificity of primary antibodies. Proc Natl Acad Sci USA, 90,1184-9.

Yokozeki, T., Ueda, H., Arai, R., Mahoney, W. and Nagamune, T. (2002) Ahomogeneous noncompetitive immunoassay for the detection of smallhaptens. Anal Chem, 74, 2500-4.

1. Non-competitive immunoassay for a small analyte comprising reacting asample containing said analyte with a reagent pair comprising a firstbinding partner that binds to said analyte, and a second binding partnerthat binds to the complex of said analyte and said first bindingpartner, wherein said second binding partner is obtained from a displayrecombinant binding partner library by selecting a binding partner thatbinds to said complex of the analyte and first binding partner, anddetermining the binding of the second binding partner, thus indicatingthe presence of the analyte in the sample.
 2. The assay of claim 1,wherein the first and second binding partners are selected from antibodyfragments Fab and scFv.
 3. The assay of claim 1, which assay is ahomogeneous assay.
 4. The assay of claim 3, which assay is based onfluorescence resonance energy transfer (FRET).
 5. The assay of claim 1,wherein the analyte is a drug of abuse.
 6. The assay of claim 5, whereinthe analyte is morphine, tetrahydrocannabinol (THC) or amphetamine. 7.Reagent pair for a non-competitive immunoassay for a small analyte,comprising a first binding partner that binds to said analyte, and asecond binding partner that binds to the complex of said analyte andsaid first binding partner, wherein said second binding partner isobtained from a display recombinant binding partner library by selectinga binding partner that binds to said complex of the analyte and firstbinding partner.
 8. Test kit for a non-competitive immunoassay for asmall analyte, said kit comprising a reagent pair comprising a firstbinding partner that binds to said analyte, and a second binding partnerthat binds to the complex of said analyte and said first bindingpartner, wherein said second binding partner is obtained from a displayrecombinant binding partner library by selecting a binding partner thatbinds to said complex of the analyte and first binding partner.
 9. Thetest kit of claim 8, wherein the first and second binding partners areselected from antibody fragments Fab and scFv.
 10. The test kit of claim8, comprising reagents for a homogeneous assay.
 11. The test kit ofclaim 10, comprising reagents for a fluorescence resonance energytransfer (FRET) based assay.
 12. The test kit of claim 8, comprisingreagents for as—saying a drug of abuse.
 13. The test kit of claim 12,comprising multiple reagent pairs for as—saying multiple drugs of abuse.14. The test kit of claim 8, comprising reagents for assaying morphine,tetrahydrocannabinol (THC) or amphetamine.
 15. The test kit of claim 14,comprising one or more reagents from the group consisting of theligand-binding portion of M1 Fab comprising SEQ ID NO 1 and SEQ ID NO 2;M2 Fab comprising SEQ ID NO 3 and SEQ ID NO 4; and K11 scFv comprisingSEQ ID NO
 5. 16. The test kit of claim 15, wherein said ligand bindingportion is formed by amino acids no. 3 to 108 of SEQ ID NO 1 and aminoacids no. 4 to 123 of SEQ ID NO 2; or of amino acids no. 3 to 108 of SEQID NO 3 and of amino acids no. 4 to 123 of SEQ ID NO 4; or of aminoacids no. 3 to 120 and no. 140 to 246 of SEQ ID NO
 5. 17. A reagent paircomprising a first binding partner that binds to ananalyte, and a secondbinding partner that binds to the complex of said analyte and said firstbinding partner, in a non-competitive immunoassay for a small analyte,whereby the second binding partner is obtained from a displayrecombinant binding partner library by selecting a binding partner thatbinds to said complex of the analyte and first binding partner. 18.Process for preparing a reagent pair for a non-competitive immunoassayfor a small analyte, comprising providing a first binding partner thatbinds to said analyte, and a second binding partner that binds to thecomplex of said analyte and said first binding partner, wherein saidsecond binding partner is obtained from a display recombinant bindingpartner library by selecting a binding partner that binds to saidcomplex of the analyte and first binding partner.
 19. The process ofclaim 18, wherein recombinant antibody fragments are prepared from aphage display library.
 20. The process of claim 18, wherein the firstbinding partner is also obtained from a display recombinant bindingpartner library.
 21. Recombinant binding protein, comprising theligand-binding portion of Ml Fab comprising SEQ ID NO 1 and SEQ ID NO 2;M2 Fab comprising SEQ ID NO 3 and SEQ ID NO 4; or K11 scFv comprisingSEQ ID NO
 5. 22. The recombinant binding protein of claim 21, whereinsaid ligand binding portion of said protein is formed by amino acids no.3 to 108 of SEQ ID NO 1 and amino acids no. 4 to 123 of SEQ ID NO 2; orof amino acids no. 3 to 108 of SEQ ID NO 3 and of amino acids no. 4 to123 of SEQ ID NO 4; or of amino acids no. 3 to 120 and no. 140 to 246 ofSEQ ID NO
 5. 23. The recombinant binding protein of claim 21, whichprotein has the amino acid sequence SEQ ID NO 1 and SEQ ID NO 2; SEQ IDNO 3 and SEQ ID NO 4; or SEQ ID NO
 5. 24. DNA, which encodes arecombinant binding protein of claim
 21. 25. Host cell, which expressesa recombinant binding protein of claim 21.